Marguerite M. Vantangoli

The Human Toxome Project

The Human Toxome Project, funded as an NIH Transformative Research grant 2011-2016, is focused on developing the concepts and the means for deducing, validating and sharing molecular pathways of toxicity (PoT). Using the test case of estrogenic endocrine disruption, the responses of MCF-7 human breast cancer cells are being phenotyped by transcriptomics and mass-spectroscopy-based metabolomics. The bioinformatics tools for PoT deduction represent a core deliverable. A number of challenges for quality and standardization of cell systems, omics technologies and bioinformatics are being addressed. In parallel, concepts for annotation, validation and sharing of PoT information, as well as their link to adverse outcomes, are being developed. A reasonably comprehensive public database of PoT, the Human Toxome Knowledge-base, could become a point of reference for toxicological research and regulatory test strategies.

Genetic variability in a frozen batch of MCF-7 cells invisible in routine authentication affecting cell function

Common recommendations for cell line authentication, annotation and quality control fall short addressing genetic heterogeneity. Within the Human Toxome Project, we demonstrate that there can be marked cellular and phenotypic heterogeneity in a single batch of the human breast adenocarcinoma cell line MCF-7 obtained directly from a cell bank that are invisible with the usual cell authentication by short tandem repeat (STR) markers. STR profiling just fulfills the purpose of authentication testing, which is to detect significant cross-contamination and cell line misidentification. Heterogeneity needs to be examined using additional methods. This heterogeneity can have serious consequences for reproducibility of experiments as shown by morphology, estrogenic growth dose-response, whole genome gene expression and untargeted mass-spectroscopy metabolomics for MCF-7 cells. Using Comparative Genomic Hybridization (CGH), differences were traced back to genetic heterogeneity already in the cells from the original frozen vials from the same ATCC lot, however, STR markers did not differ from ATCC reference for any sample. These findings underscore the need for additional quality assurance in Good Cell Culture Practice and cell characterization, especially using other methods such as CGH to reveal possible genomic heterogeneity and genetic drifts within cell lines.

MCF-7 Human Breast Cancer Cells Form Differentiated Microtissues in Scaffold-Free Hydrogels

Three-dimensional (3D) cultures are increasing in use because of their ability to represent in vivo human physiology when compared to monolayer two-dimensional (2D) cultures. When grown in 3D using scaffold-free agarose hydrogels, MCF-7 human breast cancer cells self-organize to form directionally-oriented microtissues that contain a luminal space, reminiscent of the in vivo structure of the mammary gland. When compared to MCF-7 cells cultured in 2D monolayer culture, MCF-7 microtissues exhibit increased mRNA expression of luminal epithelial markers keratin 8 and keratin 19 and decreased expression of basal marker keratin 14 and the mesenchymal marker vimentin. These 3D MCF-7 microtissues remain responsive to estrogens, as demonstrated by induction of known estrogen target mRNAs following exposure to 17β-estradiol. Culture of MCF-7 cells in scaffold-free conditions allows for the formation of more differentiated, estrogen-responsive structures that are a more relevant system for evaluation of estrogenic compounds than traditional 2D models.

Sperm mRNA Transcripts Are Indicators of Sub-Chronic Low Dose Testicular Injury in the Fischer 344 Rat

Current human reproductive risk assessment methods rely on semen and serum hormone analyses, which are not easily comparable to the histopathological endpoints and mating studies used in animal testing. Because of these limitations, there is a need to develop universal evaluations that reliably reflect male reproductive function. We hypothesized that toxicant-induced testicular injury can be detected in sperm using mRNA transcripts as indicators of insult. To test this, we exposed adult male Fischer 344 rats to low doses of model testicular toxicants and classically characterized the testicular injury while simultaneously evaluating sperm mRNA transcripts from the same animals. Overall, this study aimed to: 1) identify sperm transcripts altered after exposure to the model testicular toxicant, 2,5-hexanedione (HD) using microarrays; 2) expand on the HD-induced transcript changes in a comprehensive time course experiment using qRT-PCR arrays; and 3) test these injury indicators after exposure to another model testicular toxicant, carbendazim (CBZ). Microarray analysis of HD-treated adult Fischer 344 rats identified 128 altered sperm mRNA transcripts when compared to control using linear models of microarray analysis (q<0.05). All transcript alterations disappeared after 3 months of post-exposure recovery. In the time course experiment, time-dependent alterations were observed for 12 candidate transcripts selected from the microarray data based upon fold change and biological relevance, and 8 of these transcripts remained significantly altered after the 3-month recovery period (p<0.05). In the last experiment, 8 candidate transcripts changed after exposure to CBZ (p<0.05). The two testicular toxicants produced distinct molecular signatures with only 4 overlapping transcripts between them, each occurring in opposite directions. Overall, these results suggest that sperm mRNA transcripts are indicators of low dose toxicant-induced testicular injury in the rat.

Into the depths: Techniques for in vitro three-dimensional microtissue visualization

Three-dimensional (3-D) in vitro platforms have been shown to closely recapitulate human physiology when compared with conventional two-dimensional (2-D) in vitro or in vivo animal model systems. This confers a substantial advantage in evaluating disease mechanisms, pharmaceutical drug discovery, and toxicity testing. Despite the benefits of 3-D cell culture, limitations in visualization and imaging of 3-D microtissues present significant challenges. Here we optimized histology and microscopy techniques to overcome the constraints of 3-D imaging. For morphological assessment of 3-D microtissues of several cell types, different time points, and different sizes, a two-step glycol methacrylate embedding protocol for evaluating 3-D microtissues produced using agarose hydrogels improved resolution of nuclear and cellular histopathology characteristic of cell death and proliferation. Additional immunohistochemistry, immunofluorescence, and in situ immunostaining techniques were successfully adapted to these microtissues and enhanced by optical clearing. Utilizing the Clear(T2) protocol greatly increased fluorescence signal intensity, imaging depth, and clarity, allowing for more complete confocal fluorescence microscopy imaging of these 3-D microtissues compared with uncleared samples. The refined techniques presented here address the key challenges associated with 3-D imaging, providing new and alternative methods in evaluating disease pathogenesis, delineating toxicity pathways, and enhancing the versatility of 3-D in vitro testing systems in pharmacological and toxicological applications.

Architecture of Chimeric Spheroids Controls Drug Transport

It is well-established that upregulation of drug efflux pumps leads to multi-drug resistance. Less is known about the role of the architecture of the tumor microenvironment in this process: how the location of pump expressing cells influences drug exposure to cancerous as well as non-cancerous cells. Here, we report a 3D in vitro model of spheroids with mixtures of cells expressing high and low levels of ABCG2, quantifying pump activity by the ability to reject the fluorescent dye Hoechst 33342. With respect to the organization of the mixed spheroids, three different architectures were observed: 1) high-expressing ABCG2 cells located in the spheroid core surrounded by low-expressing cells, 2) high-expressing ABCG2 cells intermixed with low-expressing cells and 3) high-expressing ABCG2 cells surrounding a core of low-expressing cells. When high-expressing ABCG2 cells were in the core or intermixed, Hoechst uptake was directly proportional to the percentage of ABCG2 cells. When high-expressing ABCG2 cell formed an outer coating surrounding spheroids, small numbers of ABCG2 cells were disproportionately effective at inhibiting uptake. Specific inhibitors of the ABCG2 transporter eliminated the effect of this coating. Confocal microscopy of spheroids revealed the location of high- and low-expressing cells, and Hoechst fluorescence revealed that the ABCG2-dependant drug concentration in the cancer microenvironment is influenced by pump expression level and distribution among the cells within a tissue. In addition to providing a 3D model for further investigation into multicellular drug resistance, these data show that the location of ABCG2-expressing cells can control drug exposure within the tumor microenvironment.

Morphologic effects of estrogen stimulation on 3D MCF-7 microtissues

In the development of human cell-based assays, 3-dimensional (3D) cell culture models are intriguing as they are able to bridge the gap between animal models and traditional two-dimensional (2D) cell culture. Previous work has demonstrated that MCF-7 human breast carcinoma cells cultured in a 3D scaffold-free culture system self-assemble and develop into differentiated microtissues that possess a luminal space. Exposure to estradiol for 7 days decreased lumen formation in MCF-7 microtissues, altered microtissue morphology and altered expression of genes involved in estrogen signaling, cell adhesion and cell cycle regulation. Exposure to receptor-specific agonists for estrogen receptor alpha, estrogen receptor beta and g-protein coupled estrogen receptor resulted in unique, receptor-specific phenotypes and gene expression signatures. The use of a differentiated scaffold-free 3D culture system offers a unique opportunity to study the phenotypic and molecular changes associated with exposure to estrogenic compounds.

Estradiol Exposure Differentially Alters Monolayer versus Microtissue MCF-7 Human Breast Carcinoma Cultures.

The development of three-dimensional (3D) cultures is increasing, as they are able to provide the utility of in vitro models and the strength of testing in physiologically relevant systems. When cultured in a scaffold-free agarose hydrogel system, MCF-7 human breast carcinoma cells organize and develop into microtissues that contain a luminal space, in stark contrast to the flat morphology of MCF-7 two-dimensional (2D) monolayer cultures. Following exposure to 1nM E2, expression of typical estrogen-responsive genes, including progesterone receptor (PGR), PDZ containing domain 1 (PDZK1) and amphiregulin (AREG) is increased in both 2D and 3D cultures. When examining expression of other genes, particularly those involved in cell adhesion, there were large changes in 3D MCF-7 microtissues, with little to no change observed in the MCF-7 monolayer cultures. Together, these results indicate that while the initial estrogen-regulated transcriptional targets respond similarly in 2D and 3D cultures, there are large differences in activation of other pathways related to cell-cell interactions.

A 3D spheroid system to evaluate inhibitors of the ABCG2 transporter in drug uptake and penetration

None of the ABCG2 inhibitors are effective clinically against multidrug resistant tumors overexpressing ABCG2. New in vitro models are needed to characterize inhibitors and discover new ones. We report a 3D spheroid model and image-based method to quantify ABCG2 action. Nonadhesive micro-molds were used to self-assemble spheroids overexpressing ABCG2; these spheroids were then incubated with the transporter substrate Hoechst 33342. Time-lapse fluorescent microscopy was used to determine the transporter-dependent efflux of Hoechst 33342 and dose response of three inhibitors (Ko143, Iressa, Elacridar). This 3D microtissue model was also used to determine the time to maximal effect as well as duration of effect after inhibitor removal. All acted within one hour and Elacridar had a surprisingly long duration of effect, active 5 hours after removal. This model can be used with multiple cell types, provides new insight into the pharmacokinetics of inhibitors, and can be adapted to high throughput analyses.